Signaling from the mitochondrial respiratory chain is essential for acute vascular oxygen sensing in the lung.

Tripartite motif (TRIM) family proteins are post-translational protein modifiers with E3-ubiquitin ligase activity, thereby involved in various biological processes. The molecular mechanisms driving prostate cancer (PCa) bone metastasis (BM) are incompletely understood, and targetable genetic alterations are lacking in the majority of cases. Therefore, we aimed to explore the expression and potential functional relevance of 71 TRIM members in bone metastatic PCa. We performed transcriptome analysis of all human TRIM family members and 770 cancer-related genes in 29 localized PCa and 30 PCa BM using Nanostring. KEGG, STRING and Ubibrowser were used for further bioinformatic gene correlation and pathway enrichment analyses. Compared to localized tumors, six TRIMs are under-expressed while nine TRIMs are over-expressed in BM. The differentially expressed TRIM proteins are linked to TNF-, TGFβ-, PI3K/AKT- and HIF-1-signaling, and to features such as proteoglycans, platelet activation, adhesion and ECM-interaction based on correlation to cancer-related genes. The identification of TRIM-specific E3-ligase-substrates revealed insight into functional connections to oncogenes, tumor suppressors and cancer-related pathways including androgen receptor- and TGFβ signaling, cell cycle regulation and splicing. In summary, this is the first study that comprehensively and systematically characterizes the expression of all TRIM members in PCa BM. Our results describe post-translational protein modification as an important regulatory mechanism of oncogenes, tumor suppressors, and pathway molecules in PCa progression. Therefore, this study may provide evidence for novel therapeutic targets, in particular for the treatment or prevention of BM.

The pulmonary vascular tone and hypoxia-induced alterations of the pulmonary vasculature may be regulated by the mitochondrial membrane permeability transition pore (mPTP) that controls mitochondrial calcium load and apoptosis. We thus investigated, if the mitochondrial proteins p66shc and cyclophilin D (CypD) that regulate mPTP opening affect the pulmonary vascular tone. Mice deficient for p66shc (p66shc^−/−), CypD (CypD^−/−), or both proteins (p66shc/CypD^−/−) exhibited decreased pulmonary vascular resistance (PVR) compared to wild-type mice determined in isolated lungs and in vivo. In contrast, systemic arterial pressure was only lower in CypD^−/− mice. As cardiac function and pulmonary vascular remodelling did not differ between genotypes, we determined alterations of vascular contractility in isolated lungs and calcium handling in pulmonary arterial smooth muscle cells (PASMC) as underlying reason for decreased PVR. Potassium chloride (KCl)-induced pulmonary vasoconstriction and KCl-induced cytosolic calcium increase determined by Fura-2 were attenuated in all gene-deficient mice. In contrast, KCl-induced mitochondrial calcium increase determined by the genetically encoded Mito-Car-GECO and calcium retention capacity were increased only in CypD^−/− and p66shc/CypD^−/− mitochondria indicating that decreased mPTP opening affected KCl-induced intracellular calcium peaks in these cells. All mouse strains showed a similar pulmonary vascular response to chronic hypoxia, while acute hypoxic pulmonary vasoconstriction was decreased in gene-deficient mice indicating that CypD and p66shc regulate vascular contractility but not remodelling. We conclude that p66shc specifically regulates the pulmonary vascular tone, while CypD also affects systemic pressure. However, only CypD acts via regulation of mPTP opening and mitochondrial calcium regulation.

With the advance of precision medicine, the availability of tumor tissue for molecular analysis has become a limiting factor. This is particularly the case for bone metastases which are frequently occurring in cancer types such as prostate cancer. Due to the necessary decalcification process it was long thought that transcriptome analysis will not be feasible from decalcified formalin-fixed, paraffin-embedded (DFFPE) in a large manner. Here we demonstrate that mRNA extraction from DFFPE is feasible, quick, robust and reproducible and that decalcification does not hamper subsequent gene expression analysis. This might assist in implementing transcriptome analysis from DFFPE into every day practice.

Bone metastatic (BM) prostate cancer (PCa) belongs to the most lethal form of PCa, and therapeutic options are limited. Molecular profiling of metastases contributes to the understanding of mechanisms defining the bone metastatic niche. Our aim was to explore the transcriptional profile of PCa BM and to identify genes that drive progression. Paraffin-embedded tissues of 28 primary PCa and 30 BM were submitted to RNA extraction and analyzed by RNA sequencing using the Nanostring nCounter gene expression platform. A total of 770 cancer-related genes were measured using the Nanostring™ PanCancer progression panel. Gene Ontology (GO), KEGG, Reactome, STRING, Metascape, PANTHER, and Pubmed were used for data integration and gene annotation. We identified 116 differentially expressed genes (DEG) in BM compared to primaries. The most significant DEGs include CD36, FOXC2, CHAD, SPP1, MMPs, IBSP, and PTX3, which are more highly expressed in BM, and ACTG2, MYH11, CNN1, FGF2, SPOCK3, and CHRDL1, which have a lower expression. DEGs functionally relate to extracellular matrix (ECM) proteoglycans, ECM-receptors, cell-substrate adhesion, cell motility as well as receptor tyrosine kinase signaling and response to growth factors. Data integration and gene annotation of 116 DEGs were used to build a gene platform which we termed “Manually Annotated and Curated Nanostring-data Platform”. In summary, our results highlight the significance of certain genes in PCa BM to which essential pro-metastatic functions could be ascribed. Data from this study provide a comprehensive platform of genes that are related to PCa BM and provide evidence for further investigations.

Metastatic prostate cancer (mPCa) is a widespread disease with high mortality. Unraveling molecular mechanisms of disease progression is of utmost importance. The microenvironment in visceral organs and the skeletal system is of particular interest as a harbinger of metastatic spread. Therefore, we performed a comprehensive transcriptomic analysis of prostate cancer lung metastases with a special focus on differentially expressed genes attributable to the microenvironment. Digital gene expression analysis using the NanoString nCounter analysis system was performed on formalin-fixed, paraffin-embedded (FFPE) tissue from prostate cancer (PCa) lung metastases (n = 24). Data were compared to gene expression data from primary PCa and PCa bone metastases. Bioinformatic analysis was performed using several publicly available tools. In comparison to prostate cancer bone metastases, 209 genes were significantly upregulated, and 100 genes were significantly downregulated in prostate cancer lung metastases. Among the up-regulated genes, the top 10 genes with the most significant P-value were HLA-DPB1, PTPRC, ITGB7, C3, CCL21, CCL5, ITGAM, SERPINA1, MFAP4, ARAP2 and among the down-regulated genes, the top 10 genes with the most significant P-value were FOXC2, TWIST1, CDK14, CHAD, IBSP, EPN3, VIT, HAPLN1, SLC44A4, TBX1. In PCa lung metastases genes associated with immunogenic responses were upregulated while genes associated with epithelial-mesenchymal transition were down-regulated. We also showed that CXCR3/CXCL10 axis plays a significant role in prostate cancer lung metastases in comparison to bone metastases. In this study, we comprehensively explored transcriptomic alterations in PCa lung metastases in comparison to primary PCa and PCa bone metastases. In PCa lung metastases genes associated with immunogenic responses are upregulated while genes associated with epithelial-mesenchymal transition are down-regulated. This points to a more immunogenic phenotype of PCa lung metastases thus potentially making patients more susceptible to immunotherapeutic approaches.